The use of bacteria with a signal-generating metabolic activity as indicators of toxicity is well established. UK patent number GB 2005018 describes a method of assaying a liquid sample for toxic substances which involves contacting a suspension of bioluminescent microorganisms with a sample suspected of containing a toxic substance and observing the change in the light output of the bioluminescent organisms as a result of contact with the suspected toxic substance. Furthermore, a toxicity monitoring system embodying the same assay principle, which is manufactured and sold under the Trade Mark Microtox(copyright), is in routine use in both environmental laboratories and for a variety of industrial applications. An improved toxicity assay method using bioluminescent bacteria, which can be used in a wider range of test conditions than the method of GB 2005018, is described in International patent application number WO 95/10767.
The assay methods known in the prior art may utilize naturally occurring bioluminescent organisms, including Photobacterium phosphoreum and Vibrio fischeri. However, recent interest has focused on the use of genetically modified microorganisms which have been engineered to express bioluminescence. These genetically modified bioluminescent microorganisms usually express lux genes, encoding the enzyme luciferase, which have been cloned from a naturally occurring bioluminescent microorganism (E. A. Meighen (1994) Genetics of Bacterial Bioluminescence. Ann. Rev. Genet. 28: 117-139; Stewart, G. S. A. B. Jassin, S. A. A. and Denyer, S. P. (1993), Engineering Microbial bioluminescence and biosensor applications. In Molecular Diagnosis. Eds R. Rapley and M. R. Walker Blackwell Scientific Pubs/Oxford). A process for producing genetically modified bioluminescent microorganisms expressing lux genes cloned from Vibrio harveyi is described in U.S. Pat. No. 4,581,335.
The use of genetically modified bioluminescent microorganisms in toxicity testing applications has several advantages over the use of naturally occurring microorganisms. For example, it is possible to engineer microorganisms with different sensitivities to a range of different toxic substances or to a single toxic substance. However, genetically modified microorganisms are subject to marketing restrictions as a result of government legislation and there is major concern relating to the deliberate release of genetically modified microorganisms into the environment as components of commercial products. This is particularly relevant with regard to toxicity testing which is often performed in the field rather than within the laboratory. The potential risk from release of potentially pathogenic genetically modified microorganisms into the environment where they may continue to grow in an uncontrollable manner has led to the introduction of legal restrictions on the use of genetically modified organisms in the field in many countries.
It has been suggested, to avoid the problems discussed above, to use genetically modified bioluminescent microorganisms which have been treated so that they retain the metabolic function of bioluminescence but can no longer reproduce. The use of radiation (gamma-radiation), X-rays or an electron beam to kill bioluminescent cells whilst retaining the metabolic function of bioluminescence is demonstrated in International Patent Application Number WO 95/07346.
It is an object of the present invention to provide an alternative method of killing bioluminescent cells whilst retaining the metabolic function of bioluminescence which does not require the use of radiation and, as such, can be easily carried out without the need for specialized radiation equipment and containment facilities and without the risk to laboratory personnel associated with the use of radiation.
Accordingly, in a first aspect the invention provides a method of making a non-viable preparation of prokaryotic or eukaryotic cells, which preparation has a signal-generating metabolic activity, which method comprises contacting a viable culture of cells with signal-generating metabolic activity with a member of the bleomycin/phleomycin family of antibiotics.
Bleomycin and phleomycin are closely related glycopeptide antibiotics that are isolated in the form of copper chelates from cultures of Streptomyces verticillus. They represent a group of proteins with molecular weights ranging from 1000 to 10000 kda that are potent antibiotics and anti-tumour agents. So far more than 200 members of the bleomycin/phleomycin family have been isolated and characterised as complex basic glycopeptides. Family members resemble each other with respect to their physicochemical properties and their structure, indicating that functionally they all behave in the same manner. Furthermore, the chemical structure of the active moiety is conserved between family members and consists of 5 amino acids, L-glucose, 3-O-carbamoyl-D-mannose and a terminal cation. The various different bleomycin/phleomycin family members differ from each other in the nature of the terminal cation moiety, which is usually an amine. A preferred bleomycin/phleomycin antibiotic for use in the method of the invention is phleomycin D1, sold under the trade name Zeocin(trademark).
Bleomycin and phleomycin are strong, selective inhibitors of DNA synthesis in intact bacteria and in mammalian cells. Bleomycin can be observed to attack purified DNA in vitro when incubated under appropriate conditions and analysis of the bleomycin damaged DNA shows that both single-stranded and double-stranded cleavages occur, the latter being the result of staggered single strand breaks formed approximately two base pairs apart in the complementary strands.
In in vivo systems, after being taken up by the cell, bleomycin enters the cell nucleus, binds to DNA (by virtue of the interaction between its positively charged terminal amine moiety and a negatively charged phosphate group of the DNA backbone) and causes strand scission. Bleomycin causes strand scission of DNA in viruses, bacteria and eukaryotic cell systems.
The present inventors have surprisingly found that treatment of a culture of cells with signal-generating metabolic activity with a bleomycin/phleomycin antibiotic renders the culture non-viable whilst retaining a level of signal-generating metabolic activity suitable for use in toxicity testing applications. In the context of this application the term non-viable is taken to mean that the cells are unable to reproduce. The process of rendering cells non-viable whilst retaining signal-generating metabolic activity may hereinafter be referred to as xe2x80x98inactivationxe2x80x99 and cells which have been rendered non-viable according to the method of the invention may be referred to as xe2x80x98inactivatedxe2x80x99.
Because of the broad spectrum of action of the bleomycin/phleomycin family of antibiotics the method of the invention is equally applicable to bacterial cells and to eukaryotic cells with signal generating metabolic activity. Preferably the signal-generating metabolic activity is bioluminescence but other signal-generating metabolic activities which are reporters of toxic damage could be used with equivalent effect.
The method of the invention is preferred for use with bacteria or eukaryotic cells that have been genetically modified to express a signal-generating metabolic activity. The examples given below relate to E. coil which have been engineered to express bioluminescence by transformation with a plasmid carrying lux genes. The eukaryotic equivalent would be cells transfected with a vector containing nucleic acid encoding a eukaryotic luciferase enzyme (abbreviated luc) such as, for example, luciferase from the firefly Photinus pyralis. A suitable plasmid vector containing cDNA encoding firefly luciferase under the control of an SV40 viral promoter is available from Promega Corporation, Madison Wis., USA. However, in connection with the present invention it is advantageous to use recombinant cells containing the entire eukaryotic luc operon so as to avoid the need to add an exogenous substrate ( e.g. luciferin) in order to generate light output.
The optimum concentration of bleomycin/phleomycin antibiotic and contact time required to render a culture of cells non-viable whilst retaining a useful level of signal-generating metabolic activity may vary according to the cell type but can be readily determined by routine experiment. In general, the lower the concentration of antibiotic used the longer the contact time required for cell inactivation. In connection with the production of assay reagents for use in toxicity testing applications, it is generally advantageous to keep the concentration of antibiotic low (e.g. around 1-1.5 mg/ml) and increase the contact time for inactivation. As will be shown in Example 1, treatment with Zeocin(trademark) at a concentration of 1.5 mg/ml for 3 to 5 hours is sufficient to completely inactivate a culture of recombinant E. coli. 
In the case of bacteria, the contact time required to inactivate a culture of bacterial cells is found to vary according to the stage of growth of the bacterial culture at the time the antibiotic is administered. Although the method of the invention can be used on bacteria at all stages of growth it is generally preferable to perform the method on bacterial cells in an exponential growth phase because the optimum antibiotic contact time has been observed to be shortest when the antibiotic is administered to bacterial cells in an exponential growth phase.
Following treatment with bleomycin/phleomycin antibiotic the non-viable preparation of cells is preferably stabilised for ease of storage or shipment. The cells can be stabilised using known techniques such as, for example, freeze drying (lyophilization) or other cell preservation techniques known in the art. Stabilization by freeze drying has the added advantage that the freeze drying procedure itself can render cells non-viable. Thus, any cells in the preparation which remain viable after treatment of the culture with bleomycin/phleomycin antibiotic will be rendered non-viable by freeze drying. It is thought that freeze drying inactivates any remaining viable cells by enhancing the effect of antibiotic, such that sub-lethally injured cells in the culture are more sensitive to the stresses applied during freeze drying.
Prior to use the stabilised cell preparation is reconstituted using a reconstitution buffer to form an assay reagent. This reconstituted assay reagent may then be used directly in assays for analytes, for example in toxicity testing applications. It is preferable that the stabilised (i.e. freeze dried) assay reagent be reconstituted immediately prior to use, but after reconstitution it is generally necessary to allow sufficient time prior to use for the reconstituted reagent to reach a stable, high level of signal-generating activity. Suitable reconstitution buffers preferably contain an osmotically potent non-salt compound such as sucrose, dextran or polyethylene glycol, although salt based stabilisers may also be used.
Whilst the assay reagent of the invention is particularly suitable for use in toxicity testing applications it is to be understood that the invention is not limited to assay reagents for use in toxicity testing. The cell inactivation method of the invention can be used to inactivate any recombinant cells (prokaryotic or eukaryotic) with a signal generating metabolic activity that is not dependent upon cell viability.
In a further aspect the invention provides a method of assaying a potentially toxic analyte comprising the steps of,
(a) contacting a sample to be assayed for the analyte with a sample of assay reagent comprising a non-viable preparation of cells with a signal-generating metabolic activity;
(b) measuring the level of signal generated; and
(c) using the measurement obtained as an indicator of the toxicity of the analyte.
In a still further aspect, the invention provides a kit for performing the above-stated assay comprising an assay reagent with signal generating metabolic activity and means for contacting the assay reagent with a sample to be assayed for an analyte.
The analytes tested using the assay of the invention are usually toxic substances, but it is to be understood that the precise nature of the analyte to be tested is not material to the invention.
Toxicity is a general term used to describe an adverse effect on biological system and the term xe2x80x98toxic substancesxe2x80x99 includes both toxicants (synthetic chemicals that are toxic) and toxins (natural poisons). Toxicity is usually expressed as an effective concentration (EC) or inhibitory concentration (IC) value. The EC/IC value is usually denoted as a percentage response e.g. EC50, EC10 which denotes the concentration (dose) of a particular substance which affects the designated criteria for assessing toxicity (i.e. a behavioural trait or death) in the indicated proportion of the population tested. For example, an EC50 of 10 ppm indicates that 50% of the population will be affected by a concentration of 10 ppm. In the case of a toxicity assay based on the use of a bioluminescent assay reagent, the EC50 value is usually the concentration of sample substance causing a 50% change in light output.
The present invention will be further understood by way of the following Examples with reference to the accompanying Figures in which: